Intracellular ROS Induction by Ag@ZnO CoreâShell Nanoparticles

Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 24370−24381

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Intracellular ROS Induction by Ag@ZnO Core−Shell Nanoparticles: Frontiers of Permanent Optically Active Holes in Breast Cancer Theranostic Behnaz Ghaemi,† Elnaz Shaabani,†,⊥ Roqya Najafi-Taher,†,⊥ Saeedeh Jafari Nodooshan,‡ Amin Sadeghpour,§ Sharmin Kharrazi,*,† and Amir Amani*,†,∥

ACS Appl. Mater. Interfaces 2018.10:24370-24381. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 08/21/18. For personal use only.

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Department of Medical Nanotechnology, School of Advanced Technologies in Medicine (SATiM). ‡Department of Medical Biotechnology, School of Advanced Technologies in Medicine (SATiM), and ∥Medical Biomaterials Research Center (MBRC), Tehran University of Medical Sciences, 1417755469 Tehran, Iran § Centre for X-Ray Analytics, Department of Material Meet Life, Swiss Federal Laboratories for Material Science and Technology (Empa), 9014 St. Gallen, Switzerland S Supporting Information *

ABSTRACT: In this study, we investigated whether ZnO coating on Ag nanoparticles (NPs) tunes electron flux and hole figuration at the metal−semiconductor interface under UV radiation. This effect triggers the photoactivity and generation of reactive oxygen species from Ag@ZnO NPs, which results in enhanced cytotoxic effects and apoptotic cell death in human breast cancer cells (MDA-MB231). In this context, upregulation of apoptotic cascade proteins (i.e., Bax/ Bcl2 association, p53, cytochrome c, and caspase-3) along with activation of oxidative stress proteins suggested the occurrence of apoptosis by Ag@ZnO NPs in cancer cells through the mitochondrial pathway. Also, preincubation of breast cancer cells with Ag@ZnO NPs in dark conditions muted NP-related toxic effects and consequent apoptotic fate, highlighting biocompatible properties of unexcited Ag@ZnO NPs. Furthermore, the diagnostic efficacy of Ag@ZnO NPs as computed tomography (CT)/optical nanoprobes was investigated. Results confirmed the efficacy of the photoactivated system in obtaining desirable outcomes from CT/optical imaging, which represents novel theranostic NPs for simultaneous imaging and treatment of cancer. KEYWORDS: ROS generation, Ag@ZnO nanoparticle, metal−semiconductor Interfaces, optically active hole, apoptosis cascade

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INTRODUCTION Application of nanotechnology in biomedical sciences is broadly expected to change the landscape of treatment and diagnosis. While researchers are faced with tremendous challenges in controlling cell−nanoparticle (NP) interactions, novel metallic NPs have attracted important attention because of their unique interaction with biological entities.1 Advances in the development of metallic NPs are being translated into more selective and effective treatment of cancer as well as antibacterial threats.2 Among metallic nanostructures, silver NPs (Ag NPs) are widely used because of their special properties in inducing cell death mechanisms.3,4 These mechanisms help NPs to overcome multidrug resistance, resulting in the improvement of treatment efficiency.5 However, such NPs address promising therapeutic candidates, whose toxic effects are not limited to the target cells. Recent studies have revealed that the optical properties of ZnO and Ag NPs, as isolated systems, are determined by excitons and plasmons, respectively.6 In Ag@ZnO core−shell structures, a nanometric shell layer of a ZnO semiconductor © 2018 American Chemical Society

can enhance the plasmonic features of Ag with plasmon− exciton coupling.7 A direct contact between ZnO and Ag can result in interfacial processes of charge transfer, which are the most important aspects for photocatalytic reactions. Furthermore, when an external ultraviolet (UV) radiation interacts with these NPs, they can generate radicals in the environment.8 Reactive oxygen species (ROS) induction in cancer cells triggers the activation of proapoptotic enzymes for the development of apoptosis and cell death, which is more effective in photodynamic therapy with localized radiation exposure.9 A composite structure of Ag NPs, covered by ZnO as a semiconductor shell, is proposed to achieve a core−shell NP with novel and special optical/electronic and biomedical properties. A preliminary study has demonstrated special properties of the Ag@TiO2 nanostructures to collect electrons after being exposed to UV irradiation and release them in the Received: March 26, 2018 Accepted: June 21, 2018 Published: June 22, 2018 24370

DOI: 10.1021/acsami.8b03822 ACS Appl. Mater. Interfaces 2018, 10, 24370−24381

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ACS Applied Materials & Interfaces dark condition.10 Another research has shown that interfacial charge exchange between the metal and the semiconductor has a substantial role in defining the photoactivity of the composite.11 Furthermore, these metal@semiconductor core−shell clusters have photocatalytic activities and are appropriate to promote light-triggered electron-transfer reactions.10 Au@ZnO core−shell NPs showed ability for reactions based on molecules, which could be used for preparing novel optical nanocomposites for chemical sensing applications.12 Additionally, nanostructures could pave a valuable way to develop a new and versatile contrast agent for cancer imaging or tracing the target cells by multimodal theranostic agents.13 Therefore, Ag@ZnO NPs could be introduced as multifunctional NPs for the treatment of cancer (via ROS generation) while at the same time can be useful contrast agents for optical and computed tomography (CTscan) dual-mode imaging.14 In this study, Ag@ZnO NPs were synthesized and well characterized with transmission electron microscopy (TEM), UV−vis spectroscopy, X-ray diffraction (XRD), and small angle X-ray spectroscopy (SAXS). Also, release of Ag+ ions from Ag@ZnO NPs and bare Ag NPs was studied over 75 days. Breast cancer (MDA-MB231) and healthy lung fibroblast (MRC5) cells were treated with concentrations of ZnO, Ag, and Ag@ZnO NPs under UV radiation to determine cytotoxic effects and apoptosis pathways. Furthermore, intracellular ROS generation of Ag@ZnO NPs in dark condition and under UV radiation was assessed, followed by evaluation of superoxide dismutase (SOD) and glutathione reductase (GR) enzymes as a measure of cell response to oxidative stress. Moreover, mechanisms of damages caused by ROS and apoptosis pathways were assessed by the measurement of p53, Bax/ Bcl2 ratio, caspase-3, and cytochrome c. Additionally, the efficiency of Ag@ZnO NPs as a useful and versatile contrast agent for optical/CT imaging was evaluated.

Figure 1. Schematic representation of the band structure in Ag/ZnO interfaces and Fermi level balance. As the Fermi level of ZnO (Efs) is lesser than that of Ag (Efm), the electrons are transferred from Ag to the ZnO structure compensating electron equilibrium in systems with forming a new Fermi energy level (Ef) (A). Schematic shows electrontransfer process and photocatalytic activity of Ag@ZnO NPs after excitation with UV radiation. Because of the higher energy level of ZnO conduction band than the newly formed Fermi energy level of Ef, the photo-activated electrons are discharged to the metallic Ag at the interface. Then, the holes are transferred to the surface of the semiconductor to catch the electron from environmental molecules (specifically, H2O and O2) and produce ROS (B).

RESULTS AND DISCUSSION Size, Structure, and Characterization of Ag@ZnO NPs. In order to obtain an applicable multifunctional NP as a novel nanostructure with anticancer properties and imaging contrast enhancement, spherical Ag@ZnO NPs were synthesized. Figure 1 schematically illustrates the ability of the Ag@ ZnO NPs to store electrons under UV radiation and discharge them in dark. The ZnO shell coated on Ag NPs undergoes Fermi level equilibration, which cause enhancement in charge transfer after UV excitation.15 The photoinduced charge separation in the semiconductor shell is due to transfer of electrons into the metal structure (Figure 1B). The stored electrons and more likely holes are transferred to an environmental electron/hole acceptor and produce radicals.16 Moreover, when the electrons are discharged from the metal, the characteristic surface plasmon of the metal is returned.17 Figure 2A schematically represents the main steps of producing Ag@ZnO NPs. Zn+ ions are adsorbed onto the surface of negatively charged citrate-coated Ag NPs through electrostatic interactions and reacted with NaOH to allow direct formation of a ZnO shell on the surface of the Ag NP core. The synthesized NPs are calcined at 550 °C in order to obtain a better crystallization and a gradual formation of continuous ZnO shell during the annealing process.18 TEM micrograph and corresponding size distribution analysis of Ag@ZnO NPs after heat treatment show spherical morphologies with an average size of 16 nm along with a mean

thickness of 3 nm for the ZnO shell (Figures 2B,C and S1). Moreover, dynamic light scattering (DLS) studies of the dispersed NPs showed a mean size of 30 and 70 nm in water and Dulbecco’s modified Eagle’s medium (DMEM) cell culture media, respectively (Figure S2A,B). UV−vis absorption spectra of Ag NPs (∼15 nm), ZnO NPs (∼3 nm), and Ag@ ZnO (∼18 nm) NPs in the region of 200−800 nm exhibit an absorption peak at ∼420, 320, and 360 nm, respectively (Figures 2D, S3B). Absorption at 320 nm can be assigned to the absorption edge of ZnO NPs, whereas that at 420 nm can be ascribed to the characteristic surface plasmon resonance of Ag.19 After applying Mie theory for electrodynamic calculations of the Ag core and Maxwell Garnett to calculate the mean dielectric constant of the coating ZnO layer, it was possible to both qualitatively and quantitatively describe the changes in UV−vis because of the formation of an Ag@ZnO core−shell structure.20 The powder XRD patterns of ZnO NPs and calcined Ag@ZnO NPs are shown in Figure 2E from which Ag@ZnO NPs can be identified. The diffraction peaks can be assigned to the wurtzite hexagonal structure of ZnO, whereby no diffraction peak of any impurity is seen. The results show that Ag@ZnO NPs have fine crystal quality, with typical peaks of ZnO at 2-theta values. The peaks of Ag indicate strong interfacial interactions between ZnO and Ag, which is in agreement with the TEM micrographs.21 In addition, study of the XRD pattern of Ag@Zn(OH)2 has

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Figure 2. Schematic illustration of Ag@ZnO NP synthesis procedure (A). TEM micrograph of Ag@ZnO NPs (B) and corresponding size distribution analysis (C). UV−vis spectra of Ag NPs, ZnO NPs, and Ag@ZnO NPs (D). XRD spectroscopy of ZnO and Ag@ZnO NPs (E). The experimental small angle is scattered from Ag NPs and the Ag@ZnO core−shell NPs (open circles) together with the corresponding simulated curves (solid lines). The polydispersity parameters of 0.6 and 1 with a Schultz distribution function were used for Ag NPs and Ag@ZnO NPs, respectively (F). Inductively coupled plasma mass spectrometry results for release studies of Ag+ ions for 75 days, evaluated every 5 days in water solutions (G).

Figure 3. Schematic illustration of RhB-conjugated Ag@ZnO NPs (A) and zeta potential evaluation of bare NPs and RhB-conjugated NPs (B). UV−vis spectra of free Ag@ZnO NPs, RhB, and RhB-conjugated Ag@ZnO NPs (C), representative corresponding laser confocal micrograph images of MRC-5 cells (D), and MDA-MB231 (E) incubated with RhB-labeled Ag@ZnO NPs for 2 h. Scale bar: 20 μm.

shown peaks corresponding to orthogonal Zn(OH)2 (Figure S3A).

SAXS has been performed to structurally characterize the NPs in a nanometer range. The experimental scattering 24372


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Figure 4. Toxicity assessment of MRC-5 as healthy cells and MDA-MB231 as cancerous cells after treatment with different concentrations of Ag NPs, ZnO NPs, and Ag@ZnO NPs in dark condition and under UV radiation. MTT assays (A,B). LDH assays (C,D). All data were compared with control.

Ag@ZnO NPs remained stable for 75 days without any significant release of Ag+ ions. It seems that the tight crystal structure of the ZnO shell could completely prevent the release of Ag+ ions from Ag@ZnO NPs. Uptake of Ag@ZnO NPs by Cells. Confocal microscopy was used to evaluate the cellular uptake of Ag@ZnO NPs in MDA-MB231 and MRC-5 cells. Also, in order to confirm the presence of fluorescent dye on the surface of prepared RhBlabeled Ag@ZnO NPs, they were characterized by UV−vis spectroscopy as well as Zetasizer. Results of zeta potential measurement showed that conjugation of RhB on the surface of Ag@ZnO NPs reduces the electric potential of the bare surface of the Ag@ZnO NPs (Figure 3B). Interaction between surface atoms and the dye can result in the modification of surface charge, thereby changing the zeta potential.28 Moreover, the obtained results for UV−vis absorbance spectra indicate that RhB is bonded to the surface of NPs (Figure 3C).29 The spectrum exhibits an absorption peak of 367 nm for Ag@ZnO NPs and a peak of 580 nm for the RhB dye. In the case of RhB-labeled Ag@ZnO NPs, both peaks of NP and dye are noticeable because of the successful attachment of the dye to NPs.30 Additionally, labeling of Ag@ZnO NPs by RhB causes a small red shift in the absorption peak (350 nm) of Ag@ZnO NPs. Obtained results of confocal microscopy studies showed that Ag@ZnO NPs are taken up by the cells efficiently after 2 h incubation (Figures 3D,E and S4). Results showed that a substantial amount of Ag@ZnO NPs are taken up by 94% of cancer cells after 2 h of incubation, whereas NPs are taken up by 78% of healthy cells (Figure S5), which is comparable to the previous report for ZnO NPs.13 The red fluorescence of RhB in the extra nuclear region of the cells obviously shows that the Ag@ZnO NPs are internalized into the breast cancer cells (Figure 3E). The literature showed that ZnO NPs have high affinities to cancer cells.31 Also, they have different cytotoxicity profiles because of different cell uptake and cytotoxicity pathways.32 Considering size as the only determinant factor for the NP uptake, it could be suggested that the Ag@ZnO NPs have the best potent size

patterns and the corresponding simulated curves from the Ag core and the Ag@ZnO NPs are shown in Figure 2F. We have applied a polydisperse hard sphere and the polydisperse core− shell model for the simulation of scattering curves from the Ag core and the Ag@ZnO core−shell NPs, respectively. Therefore, we obtained a core size of 15.6 nm and a shell thickness of 3.0 nm. It is worth to mention that in order to simulate the scattering curves, we had to consider an asymmetric size distribution function (Schultz distribution) with quite high polydispersity, that is, 0.6 and 1.0 for the core and the core− shell particles, respectively. These findings are in good agreement with our observations from TEM images. Although there are many contradictory findings reporting the toxicity of Ag NPs, literature reveals that their cytotoxicity mainly relates to the release of Ag+ ions and ROS generation.22 Because it has been shown that the release of Ag+ ions is the main toxicity mechanism on human cells,23 it was tried to synthesize a tight shell of ZnO to minimize or eliminate the release of Ag+ ions, in addition to enhancing the photocatalytic properties of Ag@ZnO NPs.24 Therefore, evaluating the release of Ag+ ions is important for realizing their biological fate, toxicity, and biomedical impacts of Ag NPs. In this regard, release of Ag+ ions from Ag@ZnO NPs was evaluated every 5 days for a total of 75 days, and the results were compared with that of Ag NPs. Obtained results did not show any significant release of Ag+ ions from Ag@ZnO NPs, whereas 70% of Ag was released from Ag NPs, as illustrated in Figure 2G. Reports indicate that surface coating significantly determines the process kinetics of Ag+ ions in Ag NPs.25 A previous report demonstrated that 16−20% Ag+ ions were released from poly(vinylpyrrolidone) (PVP)/poly(ethylene glycol)-coated Ag NPs after 2 h, whereas the release of Ag+ ions for gelatin, citrate, and chitosan-coated Ag NPs was about 1−3% for 2 h. Thus, the role of polymer coating in controlling the release appears insignificant to act as a barrier for Ag+, which may be released afterward.26 Furthermore, polymers may quench the generation of ROS from Ag NPs and decrease the anticancer efficiency of Ag NPs.27 Results of our research indicate that 24373


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Figure 5. ROS generation ability of NPs in solution assessed with DPPH (A) and representative flow cytometry marker (B) for Ag NPs, ZnO NPs, and Ag@ZnO NP-treated MDA-MB231 cells in dark condition and under UV radiation. Intracellular H2O2 and O2• are indicated by positive DCF fluorescence and positive HE fluorescence, respectively. Representative microphotographs show a confocal study of intracellular ROS generation of Ag@ZnO NPs after UV radiation measured by DCF fluorescence in MDA-MB231 and MRC-5 cells (C). Scale bar: 10 μm.

cytotoxicity of Ag@ZnO NPs. A previous research showed that decrease of dissolution rate and amount of Ag+ ions after coating with PVP resulted in a decrease in the cytotoxicity of Ag NPs.36 Also, results of toxicity studies demonstrate that the viability of healthy cells is generally more than that of the cancerous cells after treatment with NPs. The results indicate that the healthy cells are able to tolerate mild oxidative stress because of higher antioxidant capacities.37,38 Results of the MTT assays under UV radiation have also shown a concentration-dependent reduction in the cell viability of NP-treated cell lines (Figure 4B). From the details, ZnO NPs show concentration-dependent toxicity under UV radiation in cancer cells, although with less intense effect in comparison with Ag@ZnO NPs (Figure 4B). The cytotoxicity of Ag@ZnO NPs and ZnO NPs significantly increased (80% at 30 μg/mL concentration of Ag@ZnO and 65% for ZnO NPs) in the presence of UV radiation exposure (Figure 4B). These excellent photocatalytic performances of ZnO NPs have been illustrated in previous observations.35,36 Also, our results showed that Ag NPs under UV radiation have nearly similar toxic effects in dark on both cell lines. The research demonstrated that the main toxicity mechanisms of Ag NPs are due to the release of Ag+ ions, which act as a Trojan horse, entering cells,39 a mechanism which is independent of radiation. Internalization of NPs and consequent cytotoxicity are associated with the release of LDH because of cell membrane disruption. Evaluation of LDH release showed that ZnO NPs and Ag@ZnO NPs cause ∼20% LDH release at the highest

for the internalization of cancer cells, which was reported to be about 50 nm.33 The DLS study of Ag@ZnO NPs dispersed in cell media showed a mean diameter of 70 nm. Because it was reported that 120 nm NPs are taken up through clathrinmediated endocytosis and that 50−80 nm NPs are mainly internalized by caveolin-mediated endocytosis, the synthesized NPs have the potential to be uptaken by endocytosis.34 In Vitro Toxicity Evaluation of Ag@ZnO NPs on Breast Cancer Cells. Low cytotoxicity of NPs on healthy cells is the main condition for their biomedical applications. Toxic effects of Ag NPs, ZnO NPs, and Ag@ZnO NPs on healthy lung fibroblast (MRC-5) and breast cancer (MDA-MB231) cells were assessed by 3-[4,5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase (LDH) assays in dark and under UV radiation (Figure 4A,B). MTT results in dark condition revealed a dose-dependent cytotoxicity for all types of NPs. However, the cytotoxicity of Ag NPs is significantly higher than that of the other two groups. Cell lines treated with ZnO NPs and Ag@ZnO NPs, in dark condition, remained more than 70% alive at a concentration of 20 μg/mL (Figure 4A). The findings reveal that ZnO NPs and Ag@ZnO NPs have not shown any significant cytotoxicity in cancerous and healthy cell lines in the absence of excitation radiation. A previous research showed that the natural abundance of Zn+ ions in the human body results in low toxicity of ZnO NPs, which makes it a suitable NP in biomedical applications.35 In Ag@ZnO NPs, the ZnO shell prevents the release of Ag+ ions and decreases the 24374


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Figure 6. Antioxidant response of NP-treated MDA-MB231 and MRC-5 cells in dark condition and under UV radiation, concentration of GR (A) and SOD (B) after treatment. (C) Schematic represents the cell’s oxidative stress responses initiated by SOD, GR, and glutathione peroxidase (GPx) in cells after treatment with Ag@ZnO NPs under UV radiation. The background shows the intracellular DCF green fluorescence as an indicator of ROS generation along with red-colored MD-MB 231 nucleus after treatment with Ag@ZnO NPs which were captured by confocal microscopy.

concentration (30 μg/mL) in dark conditions, whereas this value is about 80% for Ag NPs at the same concentration (Figure 4C). This result is in agreement with previous studies.40 More importantly, treatment of cells with Ag@ ZnO NPs under UV radiation significantly increases the release of LDH up to 97% in cancer cells compared with 60% for healthy cells at 30 μg/mL concentration, probably because of enhanced ROS generation in comparison with ZnO NPs. The results of LDH release indicate that the antioxidant capacity of healthy cells is sufficient for protection against a high amount of ROS.41 Also, ZnO NPs have shown 65% LDH release in both cell lines under UV radiation (Figure 4D). Furthermore, when compared both under UV radiation and in dark condition, Ag NPs have similar toxic patterns. In total, results show that Ag@ZnO NPs are highly toxic under UV radiation, whereas they are not considerably cytotoxic in dark conditions. Previous investigation has revealed that coating of biosynthesized Ag NPs by the ZnO shell has improved the antifungal properties of Ag NPs without any significant toxicity on healthy cells.42 Although this report did not consider the effect of Ag@ZnO NPs as a photocatalyst nanomaterial, the biocompatibility of synthesized NPs in dark condition was shown to improve. Therefore, these properties could make them a perfect choice in the targeted photodynamic therapy of cancer cells. Moreover, the MTT assay has been done for the toxicity assessment of the physical mixture of Ag and ZnO NPs, and the results are shown in Figure S6. Intracellular ROS-Dependent Cytotoxicity of Ag@ ZnO NPs. Ag@ZnO NPs have a high potential to generate radicals because of direct contact of ZnO and Ag, which results in interfacial processes of charge transfer when radiated by an external UV. The increased photocatalytic activity of Ag@ZnO NPs has previously been reported as a solar photocatalytic disinfection process.21,43 Also, reports showed that induction

of a high amount of ROS could remarkably overwhelm the antioxidant capacity of cells and cause cell death.44 Therefore, inspired by previous works, in this research, ROS generation was measured by the neutralization of 2,2-diphenyl1-picrylhydrazyl (DPPH) as a stable radical. Results of the DPPH assay showed that ZnO NPs and Ag@ZnO NPs generate a high amount of ROS in a concentration-dependent manner after exposure to UV radiation. Figure 5A shows that ZnO and Ag@ZnO NPs generate different amounts of ROS under UV radiation as discussed before. This result is mostly more obvious for Ag@ZnO NPs because of a higher photocatalytic activity. Previous reports have shown that ZnO NPs can produce ROS after excitation by UV light.45 Even though Ag NPs provided a strong toxicity profile in dark and under UV radiation on healthy and cancer cells, their ROS generation is less than that of the other NPs. This confirms that the main toxicity mechanism of Ag NPs is not ROS generation but most probably release of Ag+ ions.46 Moreover, the high toxicity of Ag@ZnO NPs under UV radiation is dependent on ROS generation, whereas NPs are not toxic to cells in dark condition. Ag@ZnO NPs can efficiently generate radicals during incubation with cells (e.g., mitochondrial dysfunction, Bax/Bcl2 alteration, caspase activation, and consequent apoptosis).47 The produced radicals can oxidize or reduce the DNA, lipids, proteins, and other important biological components, resulting in substantial oxidative damages to the cell. In this regard, flow cytometry was employed to evaluate different types of intracellular radicals for synthesized NPs under UV radiation (Figure 5B,C). The results of intracellular ROS in ZnO NPs and Ag@ZnO NPtreated cells under UV radiation, in respect to dark conditions, demonstrated an enhancement of dichlorofluorescein (DCF) and hydroethidine (HE) fluorescent intensity as markers of H2O2 and O2 radicals (Figure 5B). From the details, Ag@ZnO 24375


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Figure 7. Flow cytometry results of control in comparison with Ag@ZnO NP-treated cells in dark condition and under UV radiation (A). Release of Bax/Bcl2 in Ag@ZnO NP-treated cells (B). Evaluation of protein changes of cells after treatment with Ag@ZnO NPs; p53 (C), release of cytochrome c (D), and caspase-3 activity (E) in dark condition and under UV radiation. Schematic represents the apoptosis mechanisms, which are triggered by Ag@ZnO NPs. The uptaken NPs generate ROS after excitation with UV radiation, which triggers the upregulation of p53. Then, p53, as an activator of proapoptotic proteins, increases Bax and decreases the amount of Bcl2. These proteins cause mitochondrial membrane leakage with a subsequent cytochrome c release, which activates caspase-3 in the apoptotic cascade. Eventually, caspase-3 induces DNA fragmentation with the cleavage of nuclear membrane and causes apoptosis (F).

microscopy assay in MDA-MB 231 and MRC-5 cells (Figure 5C). The ROS generation capability of Ag@ZnO NPs may lead to various pathways in cells, which results in mutations and oxidative damage of cells.50,51 Also, researcher reported that ZnO NPs induce oxidative stress, with significant formation of ROS inside the cells which trigger DNA damage and eventually result in cell death.52 GR and SOD are recognized as the most important cellular antioxidant enzymes in modulation, regulation, and maintenance of cellular oxidative stress response as well as redox homoeostasis.53 Hence, to explore the effects of oxidative stress, the activity of the mentioned enzymes was monitored after treating cells with different concentrations of Ag NPs, ZnO NPs, and Ag@ZnO NPs, in dark condition and under UV radiation (Figure 6). Although ZnO NPs and Ag@ZnO NPs in dark condition did not show any significant effect on the

NPs generated higher levels of H2O2 radicals associated with permanent photoactive holes on the surface.48 These holes form spatially indirect excitons at the interface of Ag/ZnO, which transfer to the surface of NPs after UV excitation and result in a high amount of electron capture from environmental molecules and consequent H2O2.49 Ag@ZnO NPs have been reported as a high photocatalyst nanomaterial in catalysis applications and electronic procedures.15,19 Moreover, obtained results for ROS generation in dark condition (Figures 5B and S7) show no significant radical production for ZnO NPs and Ag@ZnO NP-treated cells in comparison with untreated ones. Also, the estimated results for ROS generation suggest that Ag NPs induce medium levels of H2O2 and O2 radicals in comparison with ZnO NPs and Ag@ZnO NPs. Furthermore, Ag@ZnO NP-mediated intracellular ROS generation was determined by the DCF fluorescence 24376


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Herein, an upregulation of p53 (2.5 folds) was observed at the highest concentration of Ag@ZnO NPs under UV radiation (Figure 7C). Results showed a significant difference in p53 expression between controls and cells treated with 20 and 30 μg/mL of Ag@ZnO NPs under UV radiation. Although ZnO NPs and Ag@ZnO NPs did not show any significant increase in p53 in dark condition, Ag NPs showed increase in the amount of p53 both in dark and under UV radiation. It has already been described that the Bax protein is upregulated by the p53 protein.57 Because an enhancement in Bax expression in cells treated with Ag@ZnO NP was seen, upregulation of Bax by p53 can be confirmed.60 Similar results showed that p53 plays an important role in Ag NP-induced apoptosis as a consequence of intracellular ROS generation.61 After treatment of MDA-MB231 cells with Ag@ZnO NPs under UV radiation, expression of proapoptotic Bax and p53 proteins has been upregulated, whereas expression of Bcl-2 as an antiapoptotic gene was downregulated, in comparison with control groups.59 Furthermore, release of cytochrome c is an important marker for mitochondria-mediated apoptosis. MDA-MB231 cells treated with Ag@ZnO NPs demonstrated a concentrationdependent release of cytochrome c under UV radiation. The results for ZnO NPs and Ag@ZnO NP-treated cells in dark condition have not shown any significant cytochrome release, whereas Ag NPs caused significant enhancement in the release of cytochrome c in dark condition as well as under UV radiation (Figure 7D). Also, release of cytochrome c triggers ROS production and depletion of cellular glutathione, which are related to apoptosis. Binding of cytochrome c to apoptotic protease-activating factor-1 (Apaf-1) results in an assembly of an apoptosome compound. The apoptosome could upregulate caspase-3, result in slicing substrates of aspartate residues, and consequently lead to the activation of proteolytic event in apoptosis.62 Moreover, caspase-3 is activated during apoptosis, which is known as a vital protein in initiation/execution of apoptosis. ROS is another factor that triggers the apoptotic cascade.63 In this context, activation of caspase-3 is known as a no return point in the apoptosis pathway.64 DNA cleavage is the most noticeable phenomenon in early stages of apoptosis.65 Also, caspase-3 is identified as an ultimate responsible factor for apoptosome formation in the caspase-dependent pathway of apoptosis. The results showed higher activity (2.5 fold) of caspase-3 in cells treated with 30 μg/mL of Ag@ZnO NPs under UV radiation, in comparison with controls (Figure 7E). Our apoptotic data supported the ROS-induced cytotoxicity of Ag@ZnO NPs, which causes higher apoptotic response than Ag NPs and ZnO NPs under UV radiation. Previous reports explained that one of the main toxic aspects of ZnO is due to dissolution of Zn2+ ions.66 However, some investigations state that the amount of released Zn2+ ions in aqueous medium is insufficient to stimulate the cytotoxicity in human cells.67 The schematic in Figure 7F represents the involved proteins in the apoptotic pathway, which starts with Ag@ZnO NPs as a consequence of ROS generation. Furthermore, all experiments were performed for healthy cells as well and showed that the importance of the protein expression after oxidative stress is to some extent similar in healthy and cancerous cells. CT/Optical Dual Contrast Enhancement of Ag@ZnO NPs. To evaluate possible diagnostic applications of Ag@ZnO NPs, the NPs were examined as CT/optical imaging contrast agents, followed by photoluminescence (PL) spectra evaluation.

increase of cellular antioxidant enzymes, Ag NPs significantly increased the amount of GR as well as SOD enzymes (Figure 6A,B). Increase in enzyme activity is due to intracellular ROS generation of Ag NPs in dark condition as shown by flow cytometry results. Previous reports showed that the treatment of rat liver cells with Ag NPs reduced glutathione levels and raised ROS production, representing the effect of metal NPs on the respiratory chain. Also, generated ROS in the presence of Ag NPs causes metabolic disturbances and toxicological consequences.54 Subsequently, increase in oxidative stress levels results in the enhancement of GR and SOD. After UV exposure to Ag@ZnO NPs, increased levels of SOD (10-fold) and GR (2-fold) in comparison with controls were observed. Generated oxidative stress by Ag@ZnO NPs under UV radiation as a mechanism of toxicity showed significant changes of redox balance to oxidation, which leads to oxidative damage of cells55 (Figure 6C). Distinct Cellular Fates (Apoptosis and RelatedEnzymatic Cascade). To investigate the mechanism of MDA-MB231 cell death induced by Ag@ZnO NPs, the Annexin V-PI assay was used for different concentrations. Obtained results for the highest concentration of Ag@ZnO NP-treated MDA-MB231 cells in dark condition showed 17.9% apoptosis and 5.1% necrosis, which are consistent with cytotoxicity assays (Figure 7A). The results showed 20% cell death during treatment in dark condition. However, results of UV treatment demonstrated that the main cause of cell death is apoptosis, and about 31% of the cells were positive for necrosis/late apoptosis (detailed in Figure S8). The enhanced concentration-dependent apoptosis of Ag@ZnO NPs indicates an exclusive role for ROS in killing cancerous cells.56 Participation of ROS was shown by a direct association between ROS generation and early apoptosis (Figure S8). It has also been demonstrated that induced apoptosis by Ag NPs is mediated by the oxidative stress in fibroblast, colon, and lung cancer cell lines.55,57 Reports indicated that the oxidative stress is directly involved in DNA damage and causes intrinsic mitochondriadependent apoptosis cascade. Therefore, in this study, levels of key apoptotic cascade proteins (caspase-3, Bax/Bcl2, cytochrome c, and p53) were measured in cells treated with NPs for 6 h. Among these, the B-cell lymphoma 2 (Bcl-2) family controls the permeabilization of the outer mitochondrial membrane and shows proapoptotic (Bax, Bad, Bak) effects by the activation of the internal mitochondrial permeability transition pore.58,59 Obtained results of Bax/Bcl2 studies showed no significant difference in the enhancement of Bax/ Bcl2 for cells treated with ZnO NPs and Ag@ZnO NPs in dark condition, whereas Ag NPs enhanced Bax expression and decreased Bcl2 (Figure 7B). The changes after Ag treatment may occur due to either release of Ag+ ions or ROS generation in dark condition. Moreover, the results for MDA-MB231 cells after treatment with Ag@ZnO NPs under UV radiation showed changes in Bax and Bcl2 protein folds where Bax expression was increased significantly and Bcl2 was parallelly/ correspondingly decreased. The most important role of Bcl2 is mediating the apoptosis through the mitochondrial cascade triggering the activation of released proapoptotic enzymes by the caspase pathway.47 Clearly, a large number of chemotherapeutic drugs activate the apoptosis by the activation of p53 cascade. Therefore, in this work, the effect of Ag@ZnO NP on p53 protein expression was determined using the enzyme-linked assay method. 24377


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structure of NPs makes them capable of being traced in CT imaging at potentially safe concentrations. Figure 8A shows the PL spectra of Ag@ZnO NPs in ambient temperature, which has two emission peaks in the UV−vis region with 325 nm excitation wavelength. The obtained graph shows a strong narrow peak in the UV range, positioned at 380 nm for ZnO without any specific position changes after adding Ag NPs as the core in Ag@ZnO NPs.72 Also, the lower intensity band positioned at 320 nm is related to the exciton of Ag and ZnO interfaces.73 After addition of ZnO as the shell, the PL intensity in UV is practically damped because of the ZnO/Ag charge transfer, which shows that the semiconductor−metal interface increases the photoactivity of the core−shell structure. In addition, the Ag@ZnO NPs showed a wider peak in the visible region, centered at 580 nm (i.e., 520−650) with lower intensity in comparison with pure ZnO NPs. These multicomponent wide visible emission spectra in ZnO are related to some intrinsic and extrinsic defects, which encompass the orange/red fluorescent region.74 In this regard, the results for optical images demonstrated an orange and a red fluorescent color for ZnO NPs and Ag@ZnO NPs, respectively. The fluorescent color of images turned brighter, corresponding to the rise in concentrations. Even though these NPs are not applicable for conventional confocal study, because of excitation limitation of lasers, obtained results showed that Ag@ZnO NPs could serve as effective optical imaging contrast agents. Literature shows that the noncentrosymmetric structure of ZnO NPs can be used as a nonresonant/nonlinear optical probe for biomedical applications.75 Also, previous reports demonstrate that the lanthanidedoped nanocrystals are useful fluorescent probes for bioimaging.76 Overall, these specific structures consisting of high atomic number elements in the core as well as photoactive ZnO as the shell make the NPs capable to be traced in CT/ optical imaging at safe concentrations.

In this regard, the capability of Ag@ZnO NPs as CT contrast agents was assessed by applying a 16-slice clinical CT imaging system. In order to evaluate the efficiency of synthesized NPs as CT contrast agents, their ability to attenuate X-radiation were compared with Visipaque, as a standard contrast agent of CT scan in clinical imaging. Obtained results showed an enhancement in contrast and brightness of CT images for Ag NPs and Ag@ZnO NPs, in comparison with water as negative control (Figure 8).

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CONCLUSIONS We found that Ag@ZnO NPs as the novel nanocomposite structure improve the characteristic photoactivity of ZnO NPs under UV radiation on the treatment of human breast cancer cells while represent minimum toxicity in dark conditions. The improved photocatalytic activity of Ag@ZnO NPs is related to the charge transfer at metal−semiconductor interfaces. Moreover, enhanced ROS generation and photocatalytic activity of Ag@ZnO NPs trigger apoptosis-related pathway in breast cancer cells through the activation of tumor suppressor gene p53 and along with cytochrome c and caspase-3 proteins. Also, increase in the Bax/Bcl2 ratio suggests that Ag@ZnO NPs prompt apoptosis through the mitochondrial pathway. Furthermore, Ag@ZnO NPs showed the capability to be assisted in multimodal detection by CT/optical imaging. On the basis of results, Ag@ZnO NPs are potentially applicable for simultaneous imaging and therapy as targeted photosensitizers in photodynamic therapy.

Figure 8. PL spectrum of Ag, ZnO, and Ag@ZnO NPs (A). Sixteen slice clinical CT tomographs of synthesized NPs compared with water and Visipaque. Brightness of the images indicates the signal strength (B, left), and optical images of NPs in 325 nm UV excitation in comparison with FITC as a positive fluorescent control.

Moreover, Ag@ZnO NPs showed an extensive enhancement in image contrast at concentrations of 0.08 mM compared with a conventionally used contrast agent (i.e., Visipaque at 225 mM concentration). Therefore, it is arguable that the ZnO NP protection layer in Ag@ZnO NPs makes the NPs stable and safe contrast agents for CT tomography. Additionally, the core structure of Ag@ZnO NPs makes them potent for X-ray absorption used in CT with lower concentration, compared with conventional contrast agents. The lower concentration could considerably reduce their side effects.68 Previous reports have shown that elements with high Z (atomic no.), such as lanthanides doped in the ZnO crystal, 13 Gd-coated/conjugated Au NPs,69 Ag NPs with different sizes,70 and Au−Fe alloy NPs,71 could potentially enhance the contrast of CT images. Using high atomic number elements in the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b03822. Methods and materials, TEM micrographs, DLS studies in water and DMEM, XRD pattern and UV−vis spectrum of Ag@Zn(OH)2, optical microscopy images 24378


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of Ag@ZnO NP uptake, quantitative uptake histogram, flow cytometry of intracellular ROS with dichlorodihydrofluorescein diacetate and dihydroethidium in dark condition and under UV radiation, MTT of Ag−ZnO physical mixture, and detailed amount of apoptosis/ necrosis (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.K.). *E-mail: [email protected] (A.A.). ORCID

Behnaz Ghaemi: 0000-0003-2220-0428 Elnaz Shaabani: 0000-0002-9198-4185 Roqya Najafi-Taher: 0000-0001-8468-9877 Saeedeh Jafari Nodooshan: 0000-0001-6378-7187 Amin Sadeghpour: 0000-0002-0475-7858 Sharmin Kharrazi: 0000-0001-6858-5998 Amir Amani: 0000-0003-4302-6270 Author Contributions ⊥

E.S. and R.N.-T. contributed equally in this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Tehran University of Medical Science (TUMS) through the grant number 97-01-87-37512. REFERENCES

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Intracellular ROS Induction by Ag@ZnO CoreâShell Nanoparticles

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